A Rad52 Homolog Is Required for RAD51-Independent Mitouc Recombination in Saccharom Yces Cerevisiae

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A Rad52 homolog is required for RAD51-independent mitouc recombination in Saccharom yces cerevisiae

Yun Bai and Lorraine S. Symington ~ Columbia University College of Physicians and Surgeons, Department of Microbiology and Institute of Cancer Research, New York, New York 10032 USA

With the use of an intrachromosomal inverted-repeat as a recombination reporter we have previously shown that mitotic recombination is dependent on the RAD52 gene. However, recombination was found to be reduced only 4-fold by mutation of RAD51, which encodes a homolog of bacterial RecA proteins. A rad51, which strain containing the recombination reporter was mutagenized to identify components of the RAD51-independent pathway. One mutation identified, rad59, reduced recombination 1200-fold in the presence of a rad51 mutation, but only 4- to 5-fold in a wild-type background. Thus the rad51 and rad59 mutations reduce recombination synergistically. The rad59 mutation reduced both spontaneous and double-strand-break-induced recombination between inverted repeats. However, the rate of interchromosomal recombination was increased in a rad59 homozygous diploid. These observations suggest that RAD59 functions specifically in intrachromosomal recombination. The rad59 mutant strain was sensitive to ionizing radiation, and this phenotype was used to clone the RAD59 gene by complementation. The gene encodes a protein of 238 amino acids with significant homology to members of the Rad52 family. Overexpression of RAD52 was found to suppress the DNA repair and recombination defects conferred by the rad59 mutation, suggesting that these proteins have overlapping roles or function as a complex. [Key Words: Recombination; gene conversion; RAD51; RAD52; Saccharomyces cerevisiae] Received April 26, 1996; revised version accepted July 8, 1996.

Genes in the RAD52 epistasis group (RAD50-57, XRS2, Rad52 is not homologous to any other known protein of and MRE11) were identified initially as essential for the Saccharomyces cerevisiae and has no apparent sequence repair of ionizing radiation-induced DNA damage or for motifs indicative of its function (Adzuma et al. 1984). meiosis (Game and Mortimer 1974; Ajimura et al. 1993). Homologs of Rad52 have been identified in other eu- Although the members of this family share the property karyotic organisms and show high conservation of the of conferring resistance to ionizing radiation, they show amino-terminal region (Bezzubova et al. 1993; Milne and considerable heterogeneity in other assays for double- Weaver 1993; Ostermann et al. 1993; Bendixen et al. strand break (DSB) repair and recombination. The 1994; Muris et al. 1994; Shen et al. 1995). RAD50, XRS2, and MRE11 genes form a subgroup with The yeast RAD51, RAD55, and RAD57 gene products similar properties, and interactions between these gene have sequence homology to prokaryotic RecA proteins, products have been detected by the two-hybrid system which are essential for homologous recombination and (Johzuka and Ogawa 1995). Mutation of other members the SOS response in bacteria (Kans and Mortimer 1991; of this group (RAD51, RAD52, RAD54, RAD55, and Aboussekhra et al. 1992; Basile et al. 1992; Shinohara et RAD5 7) results in the inability to repair DSBs, reduction al. 1992; Lovett 1994). Electron microscopic analysis in spontaneous and induced mitotic recombination, and shows that Rad51 forms helical filaments on both ss- defects in mating-type switching and sporulation. Of DNA and dsDNA, but only the Rad51-ssDNA nucle- these mutants, rad52 confers the most severe defects in oprotein filament is functionally relevant for strand ex- DSB repair and recombination, suggesting that RAD52 change (Ogawa et al. 1993b; Sung and Robberson 1995). plays a crucial role in these processes (Game 1993). Rad51, Rad55, and Rad57 contain conserved putative nucleotide-binding motifs called the Walker A-type and B-type boxes (Walker et al. 1982). Mutation of the conserved lysine residue within the A-motif results in mu- ~Corresponding author. tant phenotypes in DNA repair and meiosis for Rad51

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Bai and Symington and Rad55 but not for Rad57 (Shinohara et al. 1992; other HO-induced conservative recombination events Johnson and Symington 1995). can occur in the absence of RAD51, RAD54, RAD55, and Several lines of evidence suggest that the Rad proteins RAD57 {Sugawara et al. 1995; Ivanov et al. 19961. These function in a complex. The Rad51 and Rad52 proteins results suggest that an alternate pathway exists for both have been shown to interact with each other by affinity spontaneous and DSB-induced mitotic recombination chromatography (Shinohara et al. 1992) and the two-hy- events in the absence of the RAD51 pathway. To eluci- brid system {Milne and Weaver 1993; Hays et al. 1995). date the mechanism of RAD51-independent recombina- The amino-terminal region of Rad51 interacts with the tion, we searched for genes whose inactivation would carboxy-terminal region of Rad52 (Milne and Weaver further reduce recombination in the rad51 mutant back- 1993; Donovan et al. 1994). In addition, the two-hybrid ground. In this paper we describe the identification of a system has revealed interactions between Rad51 and novel gene that encodes a Rad52 homolog and show Rad54 (T. Kodadek, pets. comm.}, RadS1 and Rad55, that mutation of this gene in combination with a rad51 Rad55 and Rad57, and between Rad51 and itself (Hays et mutation synergistically reduces recombination. al. 1995; Johnson and Symington 1995). Interactions among Rad proteins are also suggested by the finding that mutations in many of these genes can be suppressed Results by overexpressing some others. For example, RAD51 or Isolation of mutants in the RAD51-independent RAD52 on a high-copy-number or CEN plasmid sup- pathway presses the DNA repair and recombination defects caused by rad55 and rad57 mutations (Hays et al. 1995; A previously described ade2 inverted-repeat substrate Johnson and Symington 1995). Overexpression of was used to measure recombination (Rattray and Syo RAD51 also suppresses certain alleles of RAD52 (Milne mington 1994)(Fig. 1). Using this substrate the recom- and Weaver 1993; Schild 1995). The large subunit of bination frequency can be visually assessed by a red/ yeast ssDNA-binding protein, encoded by the RFA1 white colony sectoring assay, and quantitated by the gene, has been found to be involved in recombination number of Ade + prototrophs within a population. The (Firmenich et al. 1995; Smith and Rothstein 1995). An average rate of recombination was determined to be 1.8 rfal missense mutation that reduces plasmid-to-chro- x 10 -4 events/cell per generation in wild-type strains mosome gene conversion and results in defects in DNA and 3.5 x 10 -s in rad51 mutants. A screen for recombi- repair is suppressed by RAD52 in a dosage-dependent nation mutants was carried out in the rad51 background. manner (Firmenich et al. 1995). Finally, both the DNA A tad51 strain {B356-13D) was mutagenized by N-me- repair and mitotic recombination defects of rad55 and thyl-N'-nitro-N-nitrosoguanidine (MNNG), and mutants rad57 null mutants are more severe at 23°C than at displaying reduced sectoring were isolated. Of -15,000 higher temperatures {Lovett and Mortimer 1987; colonies screened, five of the mutants isolated were Johnson and Symington 1995; Rattray and Sy- shown by complementation tests to be RAD52 alleles. mington 1995). This cold-sensitive phenotype suggests One other mutant, #25, displayed a low-sectoring phe- that Rad55 and Rad57 are part of, or act to stabilize, a notype similar to that of a rad52 strain. When this mu- protein complex. tant was crossed to a rad52 strain not carrying the ade2 Spontaneous mitotic recombination is generally mea- inverted-repeat substrate (LSY255), the resulting diploids sured by the rate of prototroph formation between two were resistant to ~/-ray irradiation and sectored at wild- different mutant alleles {heteroalleles) of a given gene. type levels. This result indicated: First, the low-sector- The heteroalleles may be present on homologous chro- ing phenotype of mutant #25 was not due to loss or mosomes in diploids, or as repeated sequences in hap- mutation of the inverted-repeat substrate; second, the loids. Using an intrachromosomal inverted-repeat sys- unidentified mutation in #25 was not allelic to RAD52; tem we have shown previously that spontaneous mitotic and third, the mutation was recessive. Mutant #25 was recombination is reduced more than 3000-fold in rad52 then backcrossed to an isogenic rad51 strain (B356-11A). mutants but only 4-fold in tad51 mutants (Rattray and The resulting diploids were homozygous for the rad51 Symington 1994). This result was surprising because mutation, so a RAD51-containing plasmid was intro- Rad51 is thought to be the structural and functional duced into the diploids to complement the rad51 sporu- RecA homolog in yeast, and therefore a more severe lation defect. The diploids were sporulated, and, after phenotype was expected. A rad51 rad52 double mutant tetrad dissection, plasmid-free haploid progeny were ob- showed the same rate of recombination as the rad52 sin- tained by counterselecting against the plasmid maker gle mutant, indicating that RAD52 is epistatic to gene URA3 on 5-fluoro-orotic acid (5-FOA)-containing RAD51. Similarly, RAD51 was found to be epistatic to medium. The low-sectoring phenotype segregated 2:2 in RAD54, RAD55, and RAD57 {Rattray and Symington this backcross, indicating that the unidentified mutation 1994, 1995). These results indicate that spontaneous mi- in mutant #25 was a single gene trait. Mutant #25 was totic recombination between inverted repeats is mostly derived from a rad51 strain and was thus extremely sen- dependent on RAD52, but only partially dependent on sitive to -y-ray irradiation. To test whether the unidenti- RAD51. Although mating-type switching, a double- fied mutation in #25 would confer ~/-ray sensitivity by strand-break-induced gene conversion event, is depen- itself, a strain was made that carried the unidentified dent on RAD51, RAD52, RAD54, RAD55, and RAD57, mutation but had a wild-type RAD51 gene (B357-1D).

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RAD51-independentrecombination

Figure 1. ade2 inverted-repeat substrate and ~ ade2-5'A > recombination events. The substrate contains an intrachromosomal inverted duplication of J alleles of the ade2 gene integrated at the HIS3 ~/ I AD E Z f locus on chromosome XV. One of the ade2 alleles contains a deletion on the 5' side (ade2-5'Ll), and the other one has a frame-shift mutation at a NdeI site on the 3' side (ade2- i ade2.5'A > |ade2-s'a,In~ i n). Both ade2 alleles are nonfunctional and ~sover n the initial strain {Ade-) forms red colonies on X ! nonselective media due to accumulation of a I ade2-n 1~ 0 " I ADE2 f- red pigment in the adenine biosynthetic pathway. If recombination between the two alleles produces a wild-type ADE2 gene, a white sector will be formed within the red colony. Thus the recombination level of a strain can be visually assessed by colony sectoring and quantitatively determined by measuring the ADE2 frequency of Ade + prototrophs within a pop- f ulation of cells. Ade + recombinants can arise by gene conversion and/or intrachromosomal Ade- (red) Ade+ (white) crossover events. Gene conversion restores the wild-type NdeI sequence within the full-length repeat, and crossover results in inversion of the TRP1 gene between the two ade2 alleles. The types of recombination events can be distinguished by restriction endonuclease digestion and Southern hybridization.

B357-1D displayed intermediate ~t-ray sensitivity. Based Rad59 is homologous to proteins of the Rad52 family on this property and epistasis to rad52 (see below), the A search of the protein data bases revealed that the pre- mutation in #25 was designated rad59-1 (radiation sen- dicted Rad59 protein is homologous to proteins of the sitive mutation). Rad52 family, including the Rad52 proteins from S. cerevisiae, Kluyveromyces lactis, human, mouse, chicken, Cloning of the RAD59 gene and the Rad22 protein from Schizosaccharomyces pombe (Bezzubova et al. 1993; Milne and Weaver 1993; The RAD59 gene was cloned by complementation of the Ostermann et al. 1993; Bendixen et al. 1994; Muris et al. ~-ray sensitivity of the rad59-1 mutant. Following trans- 1994; Shen et al. 1995). The Rad52 family members are formation of strain B357-1D (rad59-1) with a S. cerevi- most highly conserved at the amino terminus. Rad59 is siae genomic DNA library (Carlson and Botstein 1982) about half the length of the Rad52 family proteins and is and selection for ~-ray resistance, three independent also homologous to the amino-terminal conserved re- clones were isolated. Plasmids recovered from these gion (Fig. 2). However, Rad59 is the least conserved of transformants carried different sizes of inserts, but DNA the Rad52-1ike proteins. The Rad59 and Rad52 proteins sequence analysis revealed that all three inserts con- from S. cerevisiae have 28% residue identity and 50% tained a common DNA sequence. A subclone containing similarity. Within the most conserved region, residue a 1.2-kb ScaI-BglII fragment was capable of rescuing the identity and similarity are 37% and 55%, respectively. ~/-ray sensitivity of strain B357-1D (rad59-1) and the low- sectoring phenotype of mutant #25 (rad51 rad59-1). A DNA fragment containing the 1.2-kb ScaI-BglII fragment rad59 and rad51 synergistically reduce recombination was sequenced and an ORF encoding a predicted poly- peptide of 238 amino acids was identified. The deter- The rad59-1 mutant, like the rad51 mutant, showed mined RAD59 nucleotide sequence has been deposited only slightly reduced colony sectoring compared with into the GenBank data base under accession number the wild type. However, mutant #25 (rad51 rad59-1) was U53668. This sequence is completely identical to the isolated from a rad51 mutant background by the strong recently released sequence of chromosome IV ORF reduction in colony sectoring (Fig. 3). This synergistic D2548 in the Saccharomyces Genome Database (SGD). reduction in recombination was verified by quantita- A null allele of RAD59 (rad59::LEU2) was made by tively determining the rates of recombination for each replacing the entire coding region of RAD59 with a strain (Table 1). Compared with wild-type strains, re- LEU2 gene. A rad59::LEU2 strain (B361-4C) was then combination rates in rad51 and rad59-1 single mutants crossed to the rad59-1 strain (B357-1D), and diploids were reduced only 4- to 5-fold, whereas the rates in rad51 were found to be sporulation proficient. For tetrads with rad59-1 double mutants were reduced about 1200-fold. four viable spores, all spores were ~-ray sensitive, indi- The rad59::LEU2 null allele gave the same results as the cating that the cloned RAD59 gene was allelic to the rad59-1 allele in recombination and ~-ray sensitivity as- locus mutated in the rad59-1 mutant. says. Thus rad59-1 is likely to be a null mutation. For

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Bai and Symington

48 IGLL~KIERYTYNI~HNNKYBKHNLSK-~AL~QF~r~T~D~R ScRad59 51 SED~TK~DK~K~F~TSRIAIIINNWRV~~Q~S ScRad52 ~ K D ol~KBo K~~nNKU~F~S S RBA~WUA~Rim"JliNDNlS KIRad52 29 - - - L~SBS R~V~RBS~PIBII~F SBSRRImS WBABEmm~wmmmmmFm~mm~S SpRad22 37 - i Ki R~ MA ll|~ll|,i|'lllll-'l R~ M HsRad52 38 -iK~R~MABmqDBmm"I"a;IR~|M~ MmRad52 38 - ~ H Iln:~lhNl'illLlm Q A illlonnll'lnlm:n~vm I S lm,_lmn 14~ F ILqiIVm~l GdRad52

P " VNNGENTNTSEV ScRad59 101 T ENK sNv I n- -- R Q-- ScRad52 61 TE~JK No.,,,,T~.~- c ;r!d - - KIRad52 76 Sml R S IIINIE - SpRad22 86 I q - HsRad52 MmRad52 Figure 2. Sequence alignment of the con- GdRad52 served amino-terminal region of Rad52-1ike 147 cN~BR i T -NsNR ~ E c Y N R smnni~ K L ...... EBI IB- soR~es9 proteins. Sc: S. cerevisiae; KI: K. lactis; Sp: 99 I~T VlRN E~R~SBF R S S "6~L Yilm~l~ KIRadS2 S. pombe; Hs: H. sapiens {human); Mm: M. musculus (mouse); Gd: G. domesticus 125 MmRad52 (chicken). 125 G~K C , ~ - q GdRad52

simplification the rad59::LEU2 allele will be referred to repeat substrate, a single mutation in RAD51 or RAD59 as rad59 in subsequent analyses. Recombination rates in slightly reduced recombination on a Tn903 inverted-re- rad51 rad52 and rad52 rad59 double mutants and in peat substrate, whereas simultaneous mutation of both rad51 rad52 rad59 triple mutants were similar to that in RAD51 and RAD59 synergistically reduced recombina- rad52 single mutants, indicating that RAD52 is epistatic tion (Fig. 4A). On a leu2 inverted-repeat substrate, the to RAD51 and RAD59 with respect to the overall rate of rad59-1 mutation reduced recombination 6-fold, but the recombination. rad51 mutation had no effect. The rate of recombination With the use of the ade2 inverted-repeat recombina- in the rad51 rad59-1 double mutant was the same as that tion assay, in conjunction with Southern hybridization observed for the rad59-1 single mutant, indicating no analysis, it was shown that 50% of the Ade + prototrophs synergism between the rad51 and rad59-1 mutations on recovered from wild-type strains were noncrossover this recombination reporter (Fig. 4B). events (simple gene conversions), and the other 50% Next we tested the recombinational repair of DSBs were crossover events (simple crossovers and gene con- induced by HO endonuclease in a lacZ inverted-repeat versions with crossing-over)(Table 1). In rad51 mutants plasmid IFig. 51. A DNA fragment with an HO cut site, noncrossover events were reduced 20-fold, while cross- embedded in one copy of lacZ, serves as a site for DSB over events were reduced only 3-fold. In rad52 mutants formation when HO endonuclease is expressed. The DSB overall recombination rates were greatly reduced, but can be repaired using wild-type sequences located on a the distribution of recombination events was similar to second, promoterless, copy of lacZ on the same plasmid. wild-type strains. In rad51 rad52 double mutants the Unrepaired plasmids are lost from the population. The overall recombination rates were close to that of rad52 single mutants. However, noncrossover events in rad51 rad52 double mutants were preferentially reduced com- RAD pared with crossovers, an outcome similar to that observed in rad51 single mutants. The distribution of the classes of events in rad59 mutants is similar to that in wild-type strains but significantly different from that in radS1 strains (Table 1; P < 0.05). The Ade + prototrophs examined from rad51 rad59 double mutants show a bias rad51 -g rad59-1 toward the crossover class, but not as extreme as observed in rad51 and rad51 rad52 double mutants. This distribution of events is significantly different from 4 ! wild-type and rad59 strains (P < 0.05), but not signifi- ~, ? *;J cantly different from the distribution observed in rad51 rad51 rad59-1 strains (P > 0.05).

RAD59 is required for intrachromosomal but not in terchrom os om al recom bin a tion

To determine if the RAD59 gene has a general role in Figure 3. Colony sectoring for strains with the ade2 inverted- recombination, we tested the effect of the rad59 muta- repeat substrate. During the growth of the colony recombination on other intrachromosomal inverted repeats, on mi- tion events that generate a wild-type ADE2 gene are visualized totic interchromosomal recombination, and on spor- as white sectors within the red colony. Strains were grown at ulation. Similar to their effects on the ade2 inverted- 30°C for 7 days on YEPD medium.

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RAD51-independent recombination

B Figure 4. Recombination rates of the Tn903 eu2{ 'A~ and leu2 inverted-repeat substrates. (A) The Tn903 inverted-repeat substrate. A fragment l of the bacterial transposon Tn903 was inte- ~ IS903 leu2-5'A~ "/ grated into yeast chromosome 111 between the HIS4 and HML loci. The fragment con- Tn903 inverted repeats pRS416-SU tains the kanamycin resistance gene (kanq of Tn903, flanked by two inverted copies of the Strain genotype Rateof Kanrcells Relativerate Strain genotype Rateof Leu÷ cells Relativerate IS903 sequences. The inverted IS903 repeats can undergo an intrachromosomal reciprocal RAD 2.3 X 10-7 100 RAD 3.3 (+1.4) X 104 100 exchange resulting in inversion of the kanr rad51 5.3X 10.8 23 rad51 5.5 (_+0.5)X 104 169 gene. A yeast strain containing a single copy rad59-1 5.1 X 108 22 rad59-1 5.1 (+2.0) X 10-7 16 of the kanr gene in its original orientation is rad51 rad59-1 < 1.0 X 10-s < 4 rad51 rad59-1 4.7 (+1.3) X 10.7 14 sensitive to 0.5 mg/ml of G418. After inversion the kan ~ gene confers resistance to G418, possibly because the kanr gene is then expressed more efficiently (Willis and Klein 1987). (B) The leu2 inverted-repeat substrate. The substrate is carried on a CEN plasmid and contains two alleles of the yeast LEU2 gene that have been truncated for the 5' and 3' coding regions respectively. A functional LEU2 gene can be generated by recombination between the inverted leu2 repeats (Prado and Aguilera 1995).

efficiency of the repair process is monitored by scoring the Trp + cells similar numbers also retained the lacZ the fraction of cells that repair and thus retain the lacZ plasmid (Trp + Ura +/Trp + = 69-92%). All of the Trp + plasmid and become LacZ + after HO induction. To Ura + cells recovered were LacZ-, indicating that these avoid chromosomal cleavage of the MAT locus by the cells contained unrecombined lacZ plasmids. Eight HO endonuclease, we used strains in which the native hours after HO induction, the number of cells retaining MAT locus had been replaced by a HIS3 gene (B383-1C, the HO plasmid was still similar among all the strains -2D, -8A, 13D). These strains were cotransformed with (24-33%). The number of cells retaining both the HO the lacZ plasmid pJF5 that had a URA3 marker, and the and lacZ plasmids, however, varied significantly with plasmid pGHOT that carried a galactose-inducible HO strain background. For the wild-type strain, 44% of the endonuclease gene and a TRP1 marker. Prior to HO in- cells with the HO plasmid (Trp + ) also retained the lacZ duction, for all of the strains tested (wild-type, radS1, plasmid (Trp + Ura + ). This value decreased to 21% in the rad59, and rad51 rad59) similar numbers of cells re- rad51 strain, and to 5% in both the rad59 and the rad51 tained the HO plasmid (Trp +/Total = 50-67%), and of rad59 strains. The majority of the Trp + Ura + cells re-

Table 1. Recombination rates of the ade2 inverted-repeat substrate

Distribution of events~ Ratio of Ade ÷ rate Relative Simple gene Simple Gene conversion noncrossover Relevant genotypea ( x 10- 6)b rate conversion crossover with crossover Other d to crossoverc

RAD 180 --- 26 100 24 (50%} 17 (35%) 7 (15%) 0 1:1 rad51 35 + 4 19 3 {12%) 15 (63%) 6 {25%) 0 1:7 rad59-1 39 - 13 22 N.D. N.D. N.D. N.D. N.D. rad59 43 --- 14 24 37 (54%) 17 (25%) 14 (20%) 1 {1%) 1:1 rad51 rad59-1 0.14 --- 0.03 0.08 N.D. N.D. N.D. N.D. N.D. rad51 rad59 0.16 -+ 0.04 0.09 11 (29%) 13 {34%) 14 (37%1 0 1:2.5 rad52 0.039 -+ 0.016 0.02 16 (24%) 5 (8%) 5 (8%) 39 (60%) 1.6:1 rad51 rad52 0.050 --- 0.012 0.03 1 (4%) 6 (21%) 9 (32%) 12 (43%) 1:15 rad52 rad59 0.060 + 0.016 0.03 N.D. N.D. N.D. N.D. N.D. rad51 rad52 rad59 0.053 + 0.021 0.03 N.D. N.D. N.D. N.D. N.D. aAll mutations except rad59-1 are deletion-disruption alleles. bData from three independent experiments; rates are events/cell/generation. ¢First values indicate numbers of events recovered; in parentheses are percentages of events. Data for RAD, rad51, rad52, and rad51 rad52 strains are taken from Rattray and Symington (1994}. Strains can be divided into two groups: those not containing the rad51 mutation (RAD, rad52, rad59) and those containing the rad51 mutation (rad51, rad51 rad52, rad51 rad59). Recombination event distributions are not significantly different among strains from the same group (P > 0.05) but are significantly different between strains from different groups (P < 0.05). (N.D.) Not determined. dEvents not characterized as any of the three recombination classes. These are possibly mutagenic events. eNoncrossovers represent simple gene conversions; crossovers represent simple crossovers and gene conversions with crossover. (N.D.) Not determined.

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Bai and Symington

Before HO induction After HO induction Strain Trp÷ Trp÷ Ura+ LacZ*Trp ÷ Ura* Trp÷ Trp+ Ura+ LacZ*Trp + Ura÷ HO site genotype I total / Trp+ / Trp÷ Ura÷ I total 1Trp+ / Trp+ Ura÷ RAD 200/300 184/200 0/184 t69/566 75/169 75/75 (67%) (92%) (0%) (30%) (44%) (100%) rad51 186/300 153/186 0/153 2031619 42/203 39142 (62%) (82%) (0%) (33%) (21%) (93%) rad59 179/300 123/179 0/123 149/634 7/149 6H (60%) (69%) (0%) (24%) (5%) (86%) rad51 rad59 149/299 115/149 0/115 1881605 91188 9/9 pJF5 (50%) (77%) (0%) (31%) (5%) (100%)

Figure 5. HO-induced recombination rates of the lacZ inverted-repeat substrate. P-lacZ' represents a promoter-bearing lacZ gene whose coding region has been disrupted by an HO cut-site. PA-lacZ represents a promoterless lacZ gene with the wild-type coding sequence. In the presence of the HO endonuclease, P-lacZ' is cut at the HO site and can be repaired by gene conversion using the wild-type sequence of PA-lacZ. Unrepaired plasmids are lost from the population. Recombination was measured by scoring the number of cells retaining both the HO-producing plasmid pGHOT (Trp + ) and the lacZ plasmid pJF5 (Ura + ), and the number of Trp + Ura + cells that had generated a wild-type lacZ gene (LacZ +).

covered from each strain after HO induction were the measurable range of our assay. Thus, interchromo- LacZ +, indicating that a recombinational repair event somal recombination is dependent on RAD51 but not had taken place. These results show that the repair of RAD59. HO-induced DSBs in the lacZ inverted-repeat system is The requirement for RAD59 in meiosis was also ex- slightly dependent on RAD51 but strongly dependent on amined. Diploid strains homozygous for the rad59 mu- RAD59, and there is no synergistic effect between rad59 tation displayed 65% (65/100) sporulation efficiency and and rad51 mutations. Previously, a physical analysis us- 84% (337/400) spore viability. The corresponding rates ing the same substrate showed that a rad52 strain was in wild-type diploid strains were 78% (78/1001 and 98% completely deficient in DNA repair, and rad51, rad54, (390/400), respectively. These observations demonstrate rad55, and rad57 strains were indistinguishable from a that a rad59 mutation leads to a slight defect in meiosis. wild-type strain (Ivanov et al. 1996). The spectrum of tetrads showed no significant bias The effect of the rad59 mutation on allelic recombi- against those containing odd numbers (1 or 3) of viable nation was tested by determining the rate of Ade + pro- spores, suggesting that the meiosis defect in rad59 dip- totroph formation between ade2 alleles located on ho- loids was not due to meiosis I nondisjunction of homol- mologous chromosomes in diploids. We found that a ogous chromosomes. rad59 mutation increased the rate of spontaneous mitotic recombination between allelic ade2 sequences (Fig. 6). Rates in radS1 and rad51 rad59 strains were below rad59 is suppressed by overexpression of RAD52 To determine if the rad59 mutation can be suppressed by overexpression of RAD52, a rad59 strain was trans- ~[ ade2-n ] >~ formed with pRS416:RAD52, a single-copy-number plasmid containing the RAD52 gene, and pRS426:RAD52, a [ ] ade2-a >~ high-copy-number plasmid containing RAD52. The resulting transformants were tested for survival after ~/-ray irradiation, rad59 cells containing either single-copy- Strain genotype Rate of Ade÷ cells Relativerate number or high-copy-number RAD52 plasmids exhib- ited a similar elevation in survival compared with cells RAD/RAD 5.1 (+1.5) X 10 .7 100 containing the vector plasmids alone (Fig. 7). The eleva- rad59/rad59 3.3 (+1.7) X 10"~ 647 tion in ~-ray resistance conferred by the RAD52 plas- tad51 / rad51 < 1.0 X 10.8 < 2 mids was specific to rad59 mutant strains, since no ele- tad51 / rad51 vation was observed in wild-type strains transformed tad59 ~tad59 < 1.0 X 10.8 < 2 with these plasmids (data not shown). Notably, the RAD52 gene on a single-copy-number plasmid rescued Figure 6. Recombination rates between allelic ade2 sequences located on homologous chromosomes in diploids. One of the the ~/-ray sensitivity of the rad59 mutant to the same ade2 alleles contains a fill-in mutation at the 3' NdeI site, and extent as the RAD52 gene on a high-copy-number plas- the other one contains a fill-in mutation at the 5' AatII site mid, suggesting that a threshold level of Rad52 is re- (Huang and Symington 1994). A functional ADE2 gene can be quired for suppression. RAD52 plasmids also increased generated by interchromosomal allelic recombination. sectoring in rad51 rad59 double mutants (data not

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RADS l-independent recombination

100 domain of Rad52 (Ogawa et al. 1993a; Mortensen et al. 1996). Thus, Rad59 may have DNA binding ability as well. Overexpression of RAD52 suppresses the rad59 mutation, suggesting that Rad52 has functional redun- dancy with Rad59 or that these two proteins function in a complex. Some features of Rad59 indicate that it has functions distinct from the Rad52 protein. The carboxy- terminal region of S. cerevisiae Rad52 is responsible for the interaction with Rad51 (Milne and Weaver 1993; Donovan et al. 1994). Rad59 is only half the length of the Rad52 protein and lacks the carboxy-terminal region of Rad52 that serves as the Rad51-interacting domain. The 000'1 phenotypes caused by a rad59 mutation also differ sub- stantially from those caused by a rad52 mutation. A rad52 mutant is extremely sensitive to -?-ray irradiation, severely defective in spontaneous recombination be- 0.001 tween the ade2 inverted repeats, and completely defi- 0 20 ;0 60 80 100 120 140 cient in meiosis. A rad59 mutant shows only slight de- 7-ray dose (Krad) fects in these processes, suggesting that Rad59 and Rad52 have different roles. Figure 7. RAD52 on plasmids suppresses the ~/-ray sensitivity of rad59 mutants. Percentage survival was determined as described by Johnson and Symington (1995). (<>) rad59+pRS426; RAD59 is required for RAD51-independent (F3) rad59+pRS416; (*)rad59+pRS416:RAD59; (0)rad59+ recombination pRS426 :RAD52; (I)rad59+pRS416:RAD52. Using the ade2 intrachromosomal inverted-repeat system, recombination occurred at 20% of the wild-type level in rad51 mutants, suggesting the existence of a shown). Although overexpression of RAD51 has been largely proficient RAD51-independent recombination found to suppress certain alleles of RAD52 (Milne and mechanism. This mechanism requires the RAD59 gene Weaver 1993; Schild 1995), it was not able to suppress product, because in rad51 rad59 double mutants recom- the rad59 mutation for ~/-ray sensitivity (data not bination was reduced to about 0.1% of the wild-type shown). Furthermore, a high-copy-number plasmid con- level. Rad51, Rad55, and Rad57 are RecA homologs, taining RAD59 was unable to suppress the DNA repair and Rad51 has been shown to be a strand exchange pro- defect of a rad52 strain (data not shown}. tein (Sung 1994). Neither Rad55 nor Rad57 is required for RAD51-independent recombination, however, because the rad51 rad55 rad57 triple mutant showed the same Discussion rate of recombination as the rad51 single mutant (Rat- By searching for mutations that reduce mitotic recombi- tray and Symington 1995). Recombination rates in rad51 nation between inverted ade2 repeats in a rad51 mutant rad59 double mutants were significantly higher than background, we isolated a novel gene, RAD59, which those observed in a rad52 mutant, indicating that there encodes a Rad52 homolog. A rad59 mutation in the is a certain level of RAD52-mediated recombination in wild type background, like a rad51 mutation, reduced the absence of both RAD51 and RAD59 (Table 1). recombination between the ade2 repeats only 4- to Similar to their effects on the ade2 inverted-repeat sys- 5-fold. However, the rad59 and rad51 mutations com- tem, rad51 and rad59 mutations synergistically reduced bined synergistically reduced recombination to about recombination on the Tn903 system (Fig. 4A). For the 1200-fold below the wild-type level, rad59 mutants leu2 inverted-repeat system (Fig. 4B) and the HO-induc- showed intermediate sensitivity to ~-irradiation, and ible lacZ inverted-repeat system (Fig. 5), a rad51 muta- diploid strains homozygous for the rad59 mutation were tion had little or no effect, consistent with previous ob- slightly defective in meiosis. Overexpression of the servations (Ivanov et al. 1996). Recombination was re- RAD52 gene was able to suppress the repair and recom- duced significantly, however, by a rad59 mutation in bination phenotypes caused by a rad59 mutation. either the presence or absence of RADS1. The lack of effect by tad51 and the lack of synergism between rad51 and rad59 suggest that the RAD51 pathway is used in- Rad59 is a member of the Rad52 family efficiently, if at all, in the leu2 and lacZ systems. One The homology between the Rad59 protein and the Rad52 possible explanation for the RADS1 independence of family members suggests they may be functionally re- these two systems is that they are both plasmid-borne. It lated. Rad59 is homologous to the amino-terminal has been suggested that recombination is RAD51-inde- region of Rad52, which is conserved among all the pendent when the donor sequences for gene conversion Rad52-1ike proteins from different species (Fig. 2). The are present on a plasmid (Sugawara et al. 1995). Results amino-terminal region is thought to be the DNA-binding from all of the tested inverted-repeat systems unani-

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Bai and Symington mously show that RAD59 is required for a RAD51-inde- against noncrossovers. A rad51 mutation in rad59 and pendent recombination mechanism regardless of rad52 backgrounds also shifted the distribution of events whether it is spontaneous or HO-induced. against noncrossovers. The distribution was changed Allelic recombination was almost completely abol- from 1:1 to 1:2.5 by a rad51 mutation in the rad59 back- ished by a rad51 mutation, whereas it was elevated by a ground, and from 1.6:1 to 1:15 in the rad52 background rad59 mutation (Fig. 6). Allelic recombination and mei- (Table 1). These results suggest that the rad51 mutation, otic recombination both involve interactions between independent of RAD52 and RAD59, preferentially re- homologous chromosomes and require RAD51. RAD59 duces noncrossover compared with crossover events. is not required for allelic recombination, nor does it play Studying HO-induced recombination between lacZ di- a significant role in meiosis. One interpretation of these rect repeats, Ivanov et al. (1996) found that the gene con- results is that RAD59 is primarily involved in intrach- version product was reduced from 17% to 0.2% by a romatid and/or sister-chromatid recombination. This rad51 mutation, supporting the hypothesis that RAD51 pathway could be important for the recombinational re- is required for recombination events that are primarily pair of lesions generated during DNA replication. The noncrossovers. inactivation of RAD59 would then make substrates A rad52 mutation in both the wild-type and rad51 available for interchromosomal interactions and thus el- backgrounds showed no significant effect on the distri- evate the rate of allelic recombination. bution of events; instead, overall recombination rates were greatly reduced by the rad52 mutation (compare RAD with rad52, and rad51 with rad51 rad52 strains in The association of gene conversion with crossing-over Table 1). Similarly, a rad59 mutation also reduced over- Gene conversion (nonreciprocal) and crossing-over (re- all recombination rates in the wild-type and rad51 back- ciprocal) are the two most common types of homologous grounds, but did not change the distribution of events recombination events. In fungal meiosis, gene conver- significantly (compare RAD with rad59, and rad51 with sion events have been found to be strongly associated rad51 rad59 strains in Table 1). We suggest that Rad52 with crossing-over of flanking markers (Hurst et al. and Rad59 may form a complex at a specific stage of 1972; Petes et al. 1991). These observations have led to recombination (referred to as stage I in later discussions) the formulation of several recombination models, all of that determines the overall level of recombination. Stage which mechanistically link gene conversion with recip- I is preceded or followed by another stage (stage II) that rocal exchange (Holliday 1964; Meselson and Radding involves RAD51 and determines the distribution of re- 1975; Radding 1982; Szostak et al. 1983). Several lines of combination events. We found, however, that the rad51 evidence, however, suggest that the association of gene mutation, in addition to shifting the distribution of re- conversion with reciprocal exchange is not obligatory. combination events against noncrossovers, also influ- There are several meiotic mutants in Drosophila and enced the overall recombination rate. In the wild-type yeast in which reciprocal recombination is reduced, but and rad59 backgrounds, a rad51 mutation reduced re- gene conversion is unaffected (Carpenter 1984; Enge- combination by 5- and 270-fold respectively. Therefore, brecht et al. 1990; Sym et al. 1993; Ross-Macdonald and RAD51 also plays a role in stage I in determining the Roeder 1994). In yeast, gene conversion between allelic overall recombination rate. We suggest that in stage I sequences on homologous chromosomes is less fre- Rad51 is in the same complex with Rad52 and Rad59, quently accompanied by crossing-over in mitosis than in based on the strong evidence that Rad51 and Rad52 in- meiosis (Jinks-Robertson and Petes 1986). Intrachromo- teract with each other. Thus, Rad51 functions in two somal gene conversion between repeated sequences, in different forms. In stage I Rad51 is within the Rad52- some cases, is not tightly associated with reciprocal ex- Rad59 complex that determines the overall recombina- change (Klein and Petes 1981; Klar and Strathern 1984; tion rate, and in stage II Rad51 is outside the Rad52- Klein 1984; Jackson and Fink 1985; Aguilera and Klein Rad59 complex and promotes the process of gene con- 1989). Plasmid gap repair in Ustilago maydis, intron version unassociated with crossing-over. homing in bacteriophage T4, and repair of the DNA break resulting from P-element transposition in Pathways of mitotic recombination between Drosophila are all DSB-mediated gene conversion events inverted repeats that show a low association with crossing-over (Nassif et al. 1994; Ferguson and Holloman 1996; Mueller et al. Based on current results, we modified a previously pro- 1996). These observations suggest that mitotic recombi- posed model for pathways of mitotic recombination nation might normally occur by a mechanism that re- (Rattray and Symington 1995) and present it here as a stricts crossing-over. two-stage model (Fig. 8). Stage I determines the overall Current results support our previous proposal that level of recombination and stage II determines the dis- there is a distinct mechanism of gene conversion unas- tribution of events. In stage I in wild-type cells, a protein sociated with crossing-over, and this mechanism is de- complex containing Rad51, Rad52, Rad59, and possibly pendent on RAD51 (Rattray and Symington 1994, 1995). an unknown protein X processes recombination sub- Wild type strains displayed a 1:1 distribution for non- strates at 100% efficiency. Stage I is preceded or followed crossover and crossover events. A rad51 mutation by stage II, which consists of two alternative pathways. changed the distribution to about 1:7, strongly biased One pathway is dependent on Rad51, Rad54, Rad55, and

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RADS l-independent recombination

STAGE I STAGE II component Y and is responsible for an associated mechanism that represents the mechanisms proposed by most recombination models (Holliday 1964; Meselson and A RAD Unassociated~ __~ pathway v ~ GC Radding 1975; Szostak et al. 1983). These models suggest the existence of a recombination intermediate contain- ~ Associated ~ GC pathway ~IF- ~ ~ CO ing DNA heteroduplex and Holliday junctions. The in- (100%) GC & CO termediate is then processed by junction resolution and

B rad5l mismatch repair and gives rise to various types of products, including simple gene conversions, simple cross- ~--] Associated V7-] oc overs, and gene conversions with crossing-over. Accord- pathw,ay "- C..2..A ~ CO (2O%) GC & CO ing to this model, all of the crossover products (simple crossovers and gene conversions with crossing-over) C rad59 Unassociated~ come from the associated mechanism. Noncrossover products (simple gene conversions), on the other hand, Associated ~ GC can be derived from both the unassociated and associated pathway r ~ ~ CO (20%) GC & CO mechanisms (Fig. 8A). RAD52 and RAD59 primarily D rad51 rad59 function in stage I. Mutations in RAD52 and RAD59 @ affect overall recombination levels without having sig- ---[Associated ~ GC nificant influence on the distribution of events. RAD51 pathway r- ~ ~ CO (0.1%) " GC & CO functions in both stage I and II. A rad51 mutation reduces the overall recombination rate, as well as shifting E rad52 Unassociatedi._ the distribution of events against noncrossovers (Fig. pathv, ay r ~ GC G 8B-FI. L A,,s(iciale,.l ~ ~ GC An alternative version of this model is that the unas- padl'.',a v Y ~ ~ CO (001 ~Tr) • GC & CO sociated and associated pathways in stage II are insepa- rable in wild-type cells. Protein Y may function in the F rad51 rad52 same complex with Rad51, Rad54, Rad55, and Rad57, and this complex may mediate a single pathway gener- @ "--'~A..... ~ated ~ [~ ------~coGc pathway ating products of various types but favored for noncross- (00)%) (;C & CO overs. Rad51, Rad54, Rad55, and Rad57 in the complex Figure 8. Pathways of mitotic recombination between in- may somehow inhibit recombination intermediates verted repeats. (A) Wild-type. Recombination substrates are pro- from being resolved into crossover products. Or Rad51, cessed at 100% efficiency in stage I. The distribution of events Rad54, Rad55, and Rad57 may act to remove the invad- is determined by two alternative pathways in stage II. GC: gene ing strand after it has primed DNA repair synthesis, pre- conversion; CO: crossover; GC & CO: gene conversion accom- venting the formation of a stable Holliday junction (For- panied by crossover. (B) rad51 mutant. The stage I complex lacking Rad51 maintains 20% efficiency in processing sub- mosa and Alberts 1986). In the absence of RAD5I, which strates. In stage II the inactivation of rad51 abolishes the unas- is epistatic to RAD54, RAD55, and RAD57, protein Y sociated mechanism, resulting in a preferential decrease of non- alone leads to recombination products of relatively crossover products. The ~emaining noncrossover products come evenly distributed types. from the associated pathway. (C) rad59 mutant. RAD59 functions primarily in stage I. A rad59 mutation reduces the overall recombination rate about 5-fold without changing the distribu- Materials and methods tion of events. (D) radS1 rad59 double mutant. Simultaneous Yeast strains mutation of both RAD5I and RAD59 synergistically reduces the efficiency of the stage I complex to 0.1% of the wild-type The relevant genotypes of the yeast strains used in this study level. Noncrossovers are preferentially reduced due to the abol- are given in Table 2. All strains are derivatives of strains W303- ishment of the unassociated pathway. (E) rad52 mutant. Protein 1A or W303-1B (Thomas and Rothstein 1989). Strains contain- X in stage I mediates homologous recombination at an effi- ing the ade2-5'zl-TRPl-ade2-n construct were derived from ciency 0.01% of the wild-type level. This rate is lower than the strain B355-6D, in which the native HIS3 locus had been re- measured rate for Ade + prototroph formation, because in rad52 placed by the ade2 inverted-repeat construct, and the native mutants only a portion of the Ade + prototrophs have arisen by ADE2 locus had been replaced by the ade2::hisG-URA3-hisG recombination mechanisms (Table 1). (F) tad51 rad52 double allele (Rattray and Symington 1994). The ade2::hisG-URA3- mutant. Recombination occurs at 0.01% of the wild-type level. hisG allele can be converted into an ade2::hisG allele by select- The event distribution is like a rad51 strain. ing for URA3-deletion recombinants on medium containing 5-FOA. Strain B354-1A was constructed by one-step transplace- ment (Rothstein 1983} of strain W303-1A with a PCR fragment to generate a deletion-disruption allele of RAD51. PCR was per- Rad57, and mediates a mechanism of gene conversion formed on a HIS3-containing template DNA (pRS313) using the that is unassociated with crossing-over. Rad51, Rad54, following pair of primers: 5'-GTTACTTCTTCTATCTTC Rad55, and Rad57 possibly form a complex in this path- CGTAGTTTCCATATACTAGTAGTTGAGgtgagcgctaggagtcac- way, and RAD51 is epistatic to RAD54, RAD55, and tgc-3'; 5'-ATGTCAACCGTACTTCTCTTGCTGTTAGCAA- RAD57. The other pathway requires an unidentified AAGTATTGTTCTATcgtatagaatgatgcattacc-3'. Bases in upper

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Bai and Symington

Table 2. Yeast strains a

W303-1A MA Ta W303-1B MA Tc~ LSY255 MA Te~ rad52: :TRPI B354-1A MA Ta rad51 ::HIS3 B360-1A MA Ta rad59: :LEU2 B355-6D MA Ta ade2: :hisG-URA3-hisG his3: :ade2-5' A- TRPI -ade2-n B356-7C MA Ta ade2: :hisG his3::ade2-5'A-TRP1 -ade2-n B356-13D MA Ta ade2: :hisG his3: :ade2-5' A-TRPl-ade2-n rad51 ::HIS3 B356-11A MA Ta ade2: :hisG his3: :ade2-5' a-TRPl-ade2-n tad51 ::HIS3 #25 MATa ade2: :hisG his3::ade2-5'A-TRP1 -ade2-n radS1 ::HIS3 rad59-1 B357-1D MA Ta ade2: :hisG his3::ade2-5'A-TRP1 -ade2-n rad59-1 B361-4C MA Te~ ade2: :hisG his3::ade2-5'A-TRP1 -ade2-n rad5 9: :LEU2 B361-7D MA Te~ ade2: :hisG his3: :ade2-5' A-TRP1 -ade2-n rad5 I ::HIS3 rad59: :LE U2 B365-11C MA Ta ade2: :hisG his3: :ade2-5' A-TRP1 -ade2-n rad52: :TRP1 B365-17A MA Te~ ade2: :hisG his3:: ade2-5' A- TRPl-ade2-n rad51 ::HIS3 rad52: :TRPl B365-12C MA To~ ade2: :hisG his3:: ade2-5' A- TRP1 -ade2-n rad52: :TRPl rad59: :LE U2 B365-14B MATs ade2: :hisG his3::ade2-5'A-TRPl-ade2-n rad51::HIS3 rad52::TRP1 tad59::LEU2 U881 mat: :HIS3 pWJ554 B383-8A mat: :HIS3 B383-2D mat: :HIS3 rad51 ::HIS3 B383-13D mat::HIS3 rad59: :LE U2 B383-1C mat::HIS3 rad51 ::HIS3 rad59::LEU2 B384-1A MA Ta chromlH: :Tn903 (Tn903 = IS903-kan'-IS903) B385-10C MA Ta chromlII: :Tn903 (Tn903 = IS903-kan'-ISg03) tad51 ::HIS3 B385-4C MA Ta chromHI: :Tn903 (Tn903 = IS903-kan~-IS903) rad59-1 B385-1B MA Te~ chromlH: :Tn903 (Tn903 = IS903-kan'-IS903) rad51 ::HIS3 rad59-1 YKH 12a MA Ta ade2-n- URA3-ade2-a B366-6A MA Ta ade2-n B366-7A MA Ta ade2-a B372 MA Ta ade2-a MA To~ ade2-n B374 MATa ade2-a rad59::LEU2 MA Te~ ade2-n rad59: :LEU2 B377 MA Ta ade2-a rad51 ::HIS3 MATeL ade2-n rad51 ::HIS3 B380 MATa ade2-a rad51 ::HIS3 rad59::LE U2 MATs ade2-n rad51 ::HIS3 rad59::LEU2

aAll strains are in the W303 background (his3-11, 15 leu2-3, 112 trpl-I ura3-1 ade2-1 canl-lO0) Thomas and Rothstein 1989).

case represent RAD51 sequences and those in lower case repre- are ADE2 alleles that contain fill-in mutations at the 5' AatII sent HIS3 sequences. The resulting rad51::HIS3 allele has the and 3' NdeI sites respectively (Huang and Symington 1994). To entire RAD51 coding region replaced by the HIS3 gene. Strain generate strains B366-6A and B366-7A, intrachromosomal re- B360-1A was constructed in a similar way to generate a dele- combination events that deleted the URA3 marker of YKH12a tion-disruption allele of the RAD59 gene (rad59::LEU2) using were selected on 5-FOA medium, and red colonies (Ade-) were the following primers: 5'-GAGGGAGTCTGTGGCAGTTTA- analyzed by Southern blotting to determine which had retained GCACATGCTTTGGACCATTctcgaggagaacttctagta-3'; 5'-AT- the ade2-a or ade2-n alleles. All other strains were made by ATGCGTGCCTTTAGCATCCTCCAATTTGATAAAAGTC- mating, sporulating, and dissecting appropriate strains and Gtcgactacgtcgtaaggccg-3'. Disruptions of RAD51 and RAD59 screening for progeny with the required genotypes. were confirmed by Southern blotting or by PCR. The rad52::TRPl allele was derived from strain LSY255. Strain Plasmids B384-1A was constructed by transforming W303-1A with the A 2.8-kb ClaI-BamHI fragment containing the entire RAD59 Sad-digested pJF2 plasmid and selecting for Ura ÷ prototrophs. gene was subcloned into pRS416 to give pRS416:RAD59. In this process pJF2 was integrated into chromosome HI be- pRS416:RAD52 and pRS426:RAD52 have a 3.3-kb fragment tween HIS4 and HML loci through a 6-kb chromosome IH frag- containing the RAD52 gene cloned at the SalI site of pRS416 ment contained in the plasmid. Strain U881 was provided by J. and pRS426 respectively. YEp24:RAD51 contains a 3.7-kb Smith (Columbia University, New York, NY). In U881 the na- RAD51 fragment inserted at the BamHI site of YEp24. pJF2 tive MAT locus has been replaced by a HIS3 gene to prevent HO contains a URA3 marker, a 1.7-kb PvuII fragment of the E. coli cutting in the chromosome, and a plasmid containing the wild- transposon Tn903, and a 6-kb fragment of yeast chromosome III type MATa sequence IpWJ554) is included to allow the strain to (Willis and Klein 1987). pRS416-SU that carries the leu2 in- mate. YKH 12a contains an ade2 direct-repeat construct of ade2- verted repeats was constructed by ligating the 3.0-kb PvuI frag- n-URA3-ade2-a at the native ADE2 locus, ade2-a and ade2-n ment of pRS416 and the 5.3-kb PvuI fragment of pRS303-SU

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RADS l-independent recombination

(Prado and Aguilera 1995). pJF5 was provided by J. Haber (Bran- Determination of y-ray sensitivity deis University, Waltham, MA). pGHOT was a gift from F. Hef- Determination of the percent survival in response to ~/-irradia- fron (Oregon Health Sciences University, Portland). tion was as described by Johnson and Symington (1995).

Media, growth conditions, and genetic methods Acknowledgments Media for yeast growth were prepared as described (Sherman et al. 1986). YEPL for HO induction contains 1% (wt./vol.) yeast We thank A. Aguilera, J. Haber, F. Heffron, H. Klein, and J. extract, 2% (wt./vol.)Bacto-peptone, 3.7% (wt./vol.)lactic acid, Smith for gifts of plasmids and strains. We also thank H. Young pH 5.5. Selection for Ura- cells was performed on synthetic for helpful advice and W.K. Holloman for critical reading of the complete medium containing 5-FOA at 1 mg/ml. Selection for manuscript. This work was supported by a grant from the Na- G418 resistant cells was on YEPD medium containing 0.5 rag/ tional Institutes for Health (GM41784). ml of G418. Yeast mating, sporulation, and tetrad dissection The publication costs of this article were defrayed in part by were performed as described (Sherman et al. 1986). Cells were payment of page charges. This article must therefore be hereby grown at 30°C unless otherwise indicated. marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

Mutagenesis Mutagenesis was carried out according to Lawrence (1991). References Strain B356-13D (rad51) was grown to stationary phase at 30°C. Aboussekhra, A., R. Chanet, A. Adjiri, and F. Fabre. 1992. Semi- Cells were washed twice and resuspended in 50 mM potassium dominant suppressors of Srs2 helicase mutations of Saccha- phosphate, pH 7.0, to a density of 5 x 10 z cells/ml. Cells were romyces cerevisiae map in the RAD51 gene, whose sequence mutagenized with a final concentration of 4 mg/ml MNNG for predicts a protein with similarities to procaryotic RecA pro- 15 min at 30°C. MNNG mutagenesis was stopped by adding an teins. Mol. Cell. Biol. 12: 3224-3234. equal volume of a freshly made 10% (wt./vol.) sodium bisulfite. Adzuma, K., T. Ogawa, and H. Ogawa. 1984. Primary structure The viability of the cells after mutagenesis was -50%. Muta- of the RAD52 gene in Saccharomyces cerevisiae. Mol. Cell. tions were allowed to segregate by culturing the mutagenized Biol. 4: 2735-2744. cells in liquid YEPD for 6-8 hr at 30°C. Cells were plated on Aguilera, A. and H.L. Klein. 1989. Yeast intrachromosomal re- YEPD plates to isolate single colonies. After 7 days at 30°C, combination: Long gene conversion tracts are preferentially colonies displaying reduced sectoring were selected as candi- associated with reciprocal exchange and require the RAD1 dates for recombination mutants. and RAD3 gene products. Genetics 123" 683-694. Ajimura, M., S.H. Leem, and H. Ogawa. 1993. Identification of DNA sequencing and Sequence analysis new genes required for meiotic recombination in Saccharo- myces cerevisiae. Genetics 133:51-66. DNA sequencing was performed according to the method of Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. Sanger et al. (1977). DNA and protein data bases were searched 1990. Basic local alignment search tool. I. Mol. Biol. 215: for homology to RAD59 by means of the BLAST algorithm 403-410. (Altschul et al. 1990). Basile, G., M. Aker, and R.K. Mortimer. 1992. Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51. Mol. Cell. Biol. 12: 3235-3246. Determination of mitotic recombination rates and Bendixen, C., I. Sunjevaric, R. Bauchwitz, and R. Rothstein. characterization of recombinants 1994. Identification of a mouse homologue of the Saccharo- Determination of mitotic recombination rates between ade2 myces cerevisiae recombination and repair gene, RAD52. inverted repeats and characterization of Ade + prototrophs were Genomics 23: 300-303. as described by Rattray and Symington (1994). For each strain, Bezzubova, O.Y., H. Schmidt, K. Ostermann, W.D. Heyer, and recombination rates were measured three times on independent J.M. Buerstedde. 1993. Identification of a chicken RAD52 isolates and the mean values were presented. Recombination homologue suggests conservation of the RAD52 recombina- rates of leu2 and Tn903 inverted repeats in haploids and of ade2 tion pathway throughout the evolution of higher eukaryotes. allelic genes in diploids were measured in similar ways, except Nucleic Acids Res. 21: 5945-5949. that for the Tn903 substrate only one measurement was made. Carlson, M. and D. Botstein. 1982. Two differentially regulated HO-induced recombination was performed according to Ru- mRNAs with different 5' ends encode secreted with intra- din et al. (1989). Strains cotransformed with pJF5 and pGHOT cellular forms of yeast invertase. Celi 28: 145-154. were grown to saturation in selective medium. Cultures were Carpenter, A.T. 1984. Meiotic roles of crossing-over and of gene diluted 1:100 (vol./vol.) into YEPL and cultured overnight at conversion. Cold Spring Harbor Symp. Quant. Biol. 49: 23- 30°C to a cell density of 1 x 107 to 3 x 10 z cells/ml. Galactose 29. was added to a final concentration of 2% (wt./vol.). Cells were Donovan, J.W., G.T. Milne, and D.T. Weaver. 1994. Homotypic removed prior to and 8 hr after galactose induction, and cells and heterotypic protein associations control Rad51 function were scored for plasmid retention. LacZ + cells were identified in double-strand break repair. Genes & Dev. 8: 2552-2562. by replica-plating Trp + Ura + cells on a nitrocellulose mem- Engebrecht, J., J. Hirsch, and G.S. Roeder. 1990. Meiotic gene brane. The membrane was frozen in liquid nitrogen and thawed, conversion and crossing over: Their relationship to each then layered onto a 7-cm circle of Whatman 3MM paper soaked other and to chromosome synapsis and segregation. Cell with an indicator solution (70 mM Na2HPO4, pH 7.0, 10 mM 62: 927-937. KC1, 1 mM MgSO4, 0.27% (vol./vol.)B-mercaptoethanol, 0.02% Ferguson, D.O. and W.K. Holloman. 1996. Recombinational re- (wt./vol.) X-gal). LacZ + cells turned blue within one hour at pair of gaps in DNA is asymmetric in Ustilago maydis and 30°C. can be explained by a migrating D-loop model. Proc. Natl.

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A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae.

Y Bai and L S Symington

Genes Dev. 1996, 10: Access the most recent version at doi:10.1101/gad.10.16.2025

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